Design, Synthesis, and Evaluation of Oleyl-WRH Peptides for siRNA Delivery
The delivery of nucleic acid therapeutics across various cell membranes has always been one of the major challenges. Cell-penetrating peptides (CPPs) containing arginine (R), tryptophan (W), and histidine (H) have been promising for siRNA delivery. Therefore, to enhance siRNA delivery and efficient silencing of the STAT3 gene, herein, we hypothesized to use of fatty acylation with oleyl chain to our newly designed cell-penetrating peptide (WRH) to evaluate their silencing efficiency in breast and ovarian cancer cells. Therefore, the oleyl conjugated (WRH)n (n=1-4) peptides were synthesized using Fmoc/tBu solid-phase peptide chemistry, followed by purification using Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC), and characterization using matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy. The synthesized conjugates with a diameter of internalization, thus falling in a relatively stable range of zeta potential values (13-18 mV at N/P 40). All peptide:siRNA complexes showed an adequate binding affinity and serum stability at N/P ≥40. The peptide:siRNA complexes were found to be non-cytotoxic at N/P 40 (~20 μM) for 72 h in MDA-MB-231, MCF-7, SK-OV-3, and HEK-293 cells. Confocal microscopy and flow cytometry analysis provided qualitative and quantitative evidence for the successful cellular internalization of siRNA. Peptides oleyl-(WRH)3 and oleyl-(WRH)4 showed, respectively, ~60% and ~75% cellular uptake of siRNA in both MDA-MB-231 and SK-OV-3 cells. Western Blot data VIII for oleyl-(WRH)4 demonstrated effective silencing of the STAT-3 gene in MDA-MB-231 cells by ~75% and in SK-OV-3 cells by ~45%.
siRNA, cancer, oleic acid, cell-penetrating peptide, tryptophan, histidine, arginine, RNA interference, MDA-MB-231, SKOV-3, Western Blotting, STAT-3, Other Pharmacy and Pharmaceutical Sciences
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